Reflex principles of immunological homeostasis.

The reasoning that neural reflexes maintain homeostasis in other body organs, and that the immune system is innervated, prompted a search for neural circuits that regulate innate and adaptive immunity. This elucidated the inflammatory reflex, a prototypical reflex circuit that maintains immunological homeostasis. Molecular products of infection or injury activate sensory neurons traveling to the brainstem in the vagus nerve. The arrival of these incoming signals generates action potentials that travel from the brainstem to the spleen and other organs. This culminates in T cell release of acetylcholine, which interacts with α7 nicotinic acetylcholine receptors (α7 nAChR) on immunocompetent cells to inhibit cytokine release in macrophages. Herein is reviewed the neurophysiological basis of reflexes that provide stability to the immune system, the neural- and receptor-dependent mechanisms, and the potential opportunities for developing novel therapeutic devices and drugs that target neural pathways to treat inflammatory diseases.

Evaluation of radiolabeled acetylcholine synthesis and release in rat striatum.

Cholinergic transmission underlies higher brain functions such as cognition and movement. To elucidate the process whereby acetylcholine (ACh) release is maintained and regulated in the central nervous system, uptake of [3 H]choline and subsequent synthesis and release of [3 H]ACh were investigated in rat striatal segments. Incubation with [3 H]choline elicited efficient uptake via high-affinity choline transporter-1, resulting in accumulation of [3 H]choline and [3 H]ACh. However, following inhibition of ACh esterase (AChE), incubation with [3 H]choline led predominantly to the accumulation of [3 H]ACh. Electrical stimulation and KCl depolarization selectively released [3 H]ACh but not [3 H]choline. [3 H]ACh release gradually declined upon repetitive stimulation, whereas the release was reproducible under inhibition of AChE. [3 H]ACh release was abolished after treatment with vesamicol, an inhibitor of vesicular ACh transporter. These results suggest that releasable ACh is continually replenished from the cytosol to releasable pools of cholinergic vesicles to maintain cholinergic transmission. [3 H]ACh release evoked by electrical stimulation was abolished by tetrodotoxin, but that induced by KCl was largely resistant. ACh release was Ca2+ dependent and exhibited slightly different sensitivities to N- and P-type Ca2+ channel toxins (ω-conotoxin GVIA and ω-agatoxin IVA, respectively) between both stimuli. [3 H]ACh release was negatively regulated by M2 muscarinic and D2 dopaminergic receptors. The present results suggest that inhibition of AChE within cholinergic neurons and of presynaptic negative regulation of ACh release contributes to maintenance and facilitation of cholinergic transmission, providing a potentially useful clue for the development of therapies for cholinergic dysfunction-associated disorders, in addition to inhibition of synaptic cleft AChE.

Role of Choline in Ocular Diseases.

Choline is essential for maintaining the structure and function of cells in humans. Choline plays an important role in eye health and disease. It is a precursor of acetylcholine, a neurotransmitter of the parasympathetic nervous system, and it is involved in the production and secretion of tears by the lacrimal glands. It also contributes to the stability of the cells and tears on the ocular surface and is involved in retinal development and differentiation. Choline deficiency is associated with retinal hemorrhage, glaucoma, and dry eye syndrome. Choline supplementation may be effective for treating these diseases.

Human Neural Stem Cells Encoding ChAT Gene Restore Cognitive Function via Acetylcholine Synthesis, Aß Elimination, and Neuroregeneration in APPswe/PS1dE9 Mice.

In Alzheimer disease (AD) patients, degeneration of the cholinergic system utilizing acetylcholine for memory acquisition is observed. Since AD therapy using acetylcholinesterase (AChE) inhibitors are only palliative for memory deficits without slowing or reversing disease progress, there is a need for effective therapies, and stem cell-based therapeutic approaches targeting AD should fulfill this requirement. We established a human neural stem cell (NSC) line encoding choline acetyltransferase (ChAT) gene, an acetylcholine-synthesizing enzyme. APPswe/PS1dE9 AD model mice transplanted with the F3.ChAT NSCs exhibited improved cognitive function and physical activity. Transplanted F3.ChAT NSCs in the AD mice differentiated into neurons and astrocytes, produced ChAT protein, increased the ACh level, and improved the learning and memory function. F3.ChAT cell transplantation reduced Aß deposits by recovering microglial function; i.e., the down-regulation of ß-secretase and inflammatory cytokines and up-regulation of Aß-degrading enzyme neprilysin. F3.ChAT cells restored growth factors (GFs) and neurotrophic factors (NFs), and they induced the proliferation of NSCs in the host brain. These findings indicate that NSCs overexpressing ChAT can ameliorate complex cognitive and physical deficits of AD animals by releasing ACh, reducing Aß deposit, and promoting neuroregeneration by the production of GFs/NFs. It is suggested that NSCs overexpressing ChAT could be a candidate for cell therapy in advanced AD therapy.

A Novel Nitric Oxide Donor, S-Nitroso-NPivaloyl-D-Penicillamine, Activates a Non-Neuronal Cardiac Cholinergic System to Synthesize Acetylcholine and Augments Cardiac Function.

BACKGROUND/AIMS: In a previous study, we reported that cardiomyocytes were equipped with non-neuronal cardiac cholinergic system (NNCCS) to synthesize acetylcholine (ACh), which is indispensable for maintaining the basic physiological cardiac functions. The aim of this study was to identify and characterize a pharmacological inducer of NNCCS. METHODS: To identify a pharmacological inducer of NNCCS, we screened several chemical compounds with chemical structures similar to the structure of S-nitroso-N-acetyl-DL-penicillamine (SNAP). Preliminary investigation revealed that SNAP is an inducer of non-neuronal ACh synthesis. We screened potential pharmacological inducers in H9c2 and HEK293 cells using western blot analysis, luciferase assay, and measurements of intracellular cGMP, NO2 and ACh levels. The effects of the screened compound on cardiac function of male C57BL6 mice were also evaluated using cardiac catheter system. RESULTS: Among the tested compounds, we selected S-nitroso-Npivaloyl-D-penicillamine (SNPiP), which gradually elevated the intracellular cGMP levels and nitric oxide (NO) levels in H9c2 and HEK293 cells. These elevated levels resulted in the gradual transactivation and translation of the choline acetyltransferase gene. Additionally, in vitro and in vivo SNPiP treatment elevated ACh levels for 72 h. SNPiP-treated mice upregulated their cardiac function without tachycardia but with enhanced diastolic function resulting in improved cardiac output. The effect of SNPiP was dependent on SNPiP nitroso group as verified by the ineffectiveness of N-pivaloyl-D-penicillamine (PiP), which lacks the nitroso group. CONCLUSION: SNPiP is identified to be one of the important pharmacological candidates for induction of NNCCS.

Cigarette smoke and non-neuronal cholinergic system in the airway epithelium of COPD patients.

Acetylcholine (ACh), synthesized by Choline Acetyl-Transferase (ChAT), exerts its physiological effects via mAChRM3 in epithelial cells. We hypothesized that cigarette smoke affects ChAT, ACh, and mAChRM3 expression in the airways from COPD patients promoting airway disease. ChAT, ACh, and mAChRM3 were assessed: "ex vivo" in the epithelium from central and distal airways of COPD patients, Healthy Smoker (S) and Healthy Subjects (C), and "in vitro" in bronchial epithelial cells stimulated with cigarette smoke extract (CSE). In central airways, mAChRM3, ChAT, and ACh immunoreactivity was significantly higher in the epithelium from S and COPD than in C subjects. mAChRM3, ChAT, and ACh score of immunoreactivity was high in the metaplastia area of COPD patients. mAChRM3/ChAT and ACh/ChAT co-localization of immunoreactivity was observed in the bronchial epithelium from COPD. In vitro, CSE stimulation significantly increased mAChRM3, ChAT, and ACh expression and mAChRM3/ChAT and ACh/ChAT co-localization in 16HBE and NHBE, and increased 16HBE proliferation. Cigarette smoke modifies the levels of mAChMR3, ChAT expression, and ACh production in bronchial epithelial cells from COPD patients. Non-neuronal components of cholinergic system may have a role in the mechanism of bronchial epithelial cell proliferation, promoting alteration of normal tissue, and of related pulmonary functions.

Hippocampal Cholinergic Neurostimulating Peptide Suppresses Acetylcholine Synthesis in T Lymphocytes.

Lymphocytic cholinergic system has important roles in T cell functions, including immune responses and proliferation and differentiation of immune cells. T lymphocytes exclusively produces acetylcholine (ACh) via choline acetyltransferase (ChAT), activating their muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively) in an autocrine and paracrine manners. Hippocampal cholinergic neurostimulating peptide (HCNP) is an undecapeptide cleaved from N-terminal of phosphatidylethanolamine-binding protein 1 (PEBP1). HCNP enhances ACh synthesis through upreglation of ChAT expression in septo-hippocampal cholinergic neurons and participates in neuronal development and differentiation. Although PEBP1 and HCNP appears to be distributed ubiquitously in tissues and cells including spleen, its functions in immune cells have not been understood. In the present study, we observed that PEBP1 is also expressed in human and murine T cells. Long-term exposure to HCNP suppressed ChAT expression in MOLT3 human leukemic T cells, resulting in decreased release of ACh. HCNP also decreased the expression of extracellular signal-regulated kinase (ERK). Thus, HCNP appears to suppress lymphocytic cholinergic signaling, which might act as an immune modulator.

Possible mechanism of Vitis vinifera L. flavones on neurotransmitters, synaptic transmission and related learning and memory in Alzheimer model rats.

BACKGROUND: This study explored the possible mechanism of flavones from Vitis vinifera L. (VTF) on neurotransmitters, synaptic transmission and related learning and memory in rats with Alzheimer disease (AD). METHODS: The researchers injected amyloid-ß(25-35) into the hippocampus to establish AD model rats. The Sprague-Dawley (SD) rats were divided into a control group, a donepezil group, an AD model group, a VTF low-dose group, a VTF medium-dose group and a VTF high-dose group. The researchers detected the activity of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) according to kit instructions. The protein expression of brain-derived neurotrophic factor (BDNF), synaptotagmin-1 (SYT1) and cyclic adenosine monophosphate response element binding protein (CREB) in the rats' hippocampi was detected by immunohistochemistry and Western blot, and the gene expression of cAMP-regulated enhancer (CRE) was detected by real-time quantitative polymerase chain reaction (PCR). RESULTS: VTF may enhance the protein expression of p-CREB, BDNF and SYT1 in rat hippocampi, depending on dose. The messenger RNA (mRNA) level of CREB was significantly higher in the VTF high-dose group than in the model group, which was consistent with the results of Western blotting. VTF may reduce the activity of AChE and increase that of ChAT in rat hippocampi. Finally, VTF effectively improved the learning and memory abilities of AD rats. CONCLUSIONS: VTF can promote synaptic plasticity and indirectly affect the expression of cholinergic neurotransmitters, which may be one mechanism of VTF protection in AD rats.

Acetylcholine induces fibrogenic effects via M2/M3 acetylcholine receptors in non-alcoholic steatohepatitis and in primary human hepatic stellate cells.

BACKGROUND: The parasympathetic nervous system (PNS), via neurotransmitter acetylcholine (ACh), modulates fibrogenesis in animal models. However, the role of ACh in human hepatic fibrogenesis is unclear. AIMS: We aimed to determine the fibrogenic responses of human hepatic stellate cells (hHSC) to ACh and the relevance of the PNS in hepatic fibrosis in patients with non-alcoholic steatohepatitis (NASH). METHODS: Primary hHSC were analyzed for synthesis of endogenous ACh and acetylcholinesterase and gene expression of choline acetyltransferase and muscarinic ACh receptors (mAChR). Cell proliferation and fibrogenic markers were analyzed in hHSC exposed to ACh, atropine, mecamylamine, methoctramine, and 4-diphenylacetoxy-N-methylpiperidine methiodide. mAChR expression was analyzed in human NASH scored for fibrosis. RESULTS: We observed that hHSC synthesize ACh and acetylcholinesterase and express choline acetyltransferase and M1-M5 mAChR. We also show that M2 was increased during NASH progression, while both M2 and M3 were found upregulated in activated hHSC. Furthermore, endogenous ACh is required for hHSC basal growth. Exogenous ACh resulted in hHSC hyperproliferation via mAChR and phosphoinositide 3-kinase and Mitogen-activated protein kinase kinase (MEK) signaling pathways, as well as increased fibrogenic markers. CONCLUSION: We show that ACh regulates hHSC activation via M2 and M3 mAChR involving the phosphoinositide 3-kinase and MEK pathways in vitro. Finally, we provide evidence that the PNS may be involved in human NASH fibrosis.

Lymphocyte-derived ACh regulates local innate but not adaptive immunity.

Appropriate control of immune responses is a critical determinant of health. Here, we show that choline acetyltransferase (ChAT) is expressed and ACh is produced by B cells and other immune cells that have an impact on innate immunity. ChAT expression occurs in mucosal-associated lymph tissue, subsequent to microbial colonization, and is reduced by antibiotic treatment. MyD88-dependent Toll-like receptor up-regulates ChAT in a transient manner. Unlike the previously described CD4(+) T-cell population that is stimulated by norepinephrine to release ACh, ChAT(+) B cells release ACh after stimulation with sulfated cholecystokinin but not norepinephrine. ACh-producing B-cells reduce peritoneal neutrophil recruitment during sterile endotoxemia independent of the vagus nerve, without affecting innate immune cell activation. Endothelial cells treated with ACh in vitro reduced endothelial cell adhesion molecule expression in a muscarinic receptor-dependent manner. Despite this ability, ChAT(+) B cells were unable to suppress effector T-cell function in vivo. Therefore, ACh produced by lymphocytes has specific functions, with ChAT(+) B cells controlling the local recruitment of neutrophils.

Ginsenoside Re and Rd enhance the expression of cholinergic markers and neuronal differentiation in Neuro-2a cells.

In Alzheimer's disease (AD), extensive neuronal loss and a deficiency of the neurotransmitter acetylcholine (ACh) are the major characteristics during pathogenesis in the brain. In the present study, we aimed to investigate whether representative ginsenosides from ginseng can regulate choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT), which are required for cholinergic neurotransmission. Our results revealed that Re and Rd induced effectively the expression of ChAT/VAChT genes in Neuro-2a cells as well as ACh elevation. Microtubule-associated protein-2 (MAP-2), nerve growth factor receptor (p75), p21, and TrkA genes and proteins were also significantly expressed. Moreover, both activated extracelullar signal-regulated protein kinase (ERK) and Akt were inhibited by K252a, a selective Trk receptor inhibitor. These findings strongly indicate that Re and Rd play an important role in neuronal differentiation and the nerve growth factor (NGF)-TrkA signaling pathway. High performance liquid chromatography analysis showed that Re and Rd administered orally were transported successfully into brain tissue and increased the level of ChAT and VAChT mRNA. The present study demonstrates that Re and Rd are selective candidates for upregulation of the expression of cholinergic markers, which may counter the symptoms and progress of AD.

Bacterial neuroactive compounds produced by psychobiotics.

We recently coined the phrase 'psychobiotics' to describe an emerging class of probiotics of relevance to psychiatry [Dinan et al., Biol Psychiatry 2013;74(10):720-726]. Such "mind-altering" probiotics may act via their ability to produce various biologically active compounds, such as peptides and mediators normally associated with mammalian neurotransmission. Several molecules with neuroactive functions such as gamma-aminobutyric acid (GABA), serotonin, catecholamines and acetylcholine have been reported to be microbially-derived, many of which have been isolated from bacteria within the human gut. Secreted neurotransmitters from bacteria in the intestinal lumen may induce epithelial cells to release molecules that in turn modulate neural signalling within the enteric nervous system and consequently signal brain function and behaviour of the host. Consequently, neurochemical containing/producing probiotic bacteria may be viewed as delivery vehicles for neuroactive compounds and as such, probiotic bacteria may possibly have the potential as a therapeutic strategy in the prevention and/or treatment of certain neurological and neurophysiological conditions.

Development of NMR spectroscopic methods for dynamic detection of acetylcholine synthesis by choline acetyltransferase in hippocampal tissue.

Choline acetyltransferase (ChAT) is the key enzyme for acetylcholine (ACh) synthesis and constitutes a reliable marker for the integrity of cholinergic neurons. Cortical ChAT activity is decreased in the brain of patients suffering from Alzheimer's and Parkinson's diseases. The standard method used to measure the activity of ChAT enzyme relies on a very sensitive radiometric assay, but can only be performed on post-mortem tissue samples. Here, we demonstrate the possibility to monitor ACh synthesis in rat brain homogenates in real time using NMR spectroscopy. First, the experimental conditions of the radiometric assay were carefully adjusted to produce maximum ACh levels. This was important for translating the assay to NMR, which has a low intrinsic sensitivity. We then used (15) N-choline and a pulse sequence designed to filter proton polarization by nitrogen coupling before (1) H-NMR detection. ACh signal was resolved from choline signal and therefore it was possible to monitor ChAT-mediated ACh synthesis selectively over time. We propose that the present approach using a labeled precursor to monitor the enzymatic synthesis of ACh in rat brain homogenates through real-time NMR represents a useful tool to detect neurotransmitter synthesis. This method may be adapted to assess the state of the cholinergic system in the brain in vivo in a non-invasive manner using NMR spectroscopic techniques.

Choline transporter-like protein 4 (CTL4) links to non-neuronal acetylcholine synthesis.

Synthesis of acetylcholine (ACh) by non-neuronal cells is now well established and plays diverse physiologic roles. In neurons, the Na(+) -dependent, high affinity choline transporter (CHT1) is absolutely required for ACh synthesis. In contrast, some non-neuronal cells synthesize ACh in the absence of CHT1 indicating a fundamental difference in ACh synthesis compared to neurons. The aim of this study was to identify choline transporters, other than CHT1, that play a role in non-neuronal ACh synthesis. ACh synthesis was studied in lung and colon cancer cell lines focusing on the choline transporter-like proteins, a five gene family choline-transporter like protein (CTL)1-5. Supporting a role for CTLs in choline transport in lung cancer cells, choline transport was Na(+) -independent and CTL1-5 were expressed in all cells examined. CTL1, 2, and 5 were expressed at highest levels and knockdown of CTL1, 2, and 5 decreased choline transport in H82 lung cancer cells. Knockdowns of CTL1, 2, 3, and 5 had no effect on ACh synthesis in H82 cells. In contrast, knockdown of CTL4 significantly decreased ACh secretion by both lung and colon cancer cells. Conversely, increasing expression of CTL4 increased ACh secretion. These results indicate that CTL4 mediates ACh synthesis in non-neuronal cell lines and presents a mechanism to target non-neuronal ACh synthesis without affecting neuronal ACh synthesis.

Memory-enhancing effect of Mori Fructus via induction of nerve growth factor.

Fruits rich in phytochemicals have been shown to improve memory by protecting or enhancing neuronal functions mediated by neurotrophic factors, such as nerve growth factor (NGF), in the hippocampus. Mori Fructus (Morus alba L., Moraceae), also called mulberry, is used as a food, dietary supplement and an anti-ageing agent in traditional Oriental medicine. It is also known to contain abundant flavonoid compounds and to exhibit various pharmacological effects. The present study was performed to evaluate the memory-enhancing effect of Mori Fructus extract (ME) in mice, with a focus on NGF regulation. ME (20, 100 and 500 mg/kg per d for 7 d, per os) dose-dependently promoted NGF release in the mouse hippocampus, leading to phosphorylation of extracellular signal-regulated kinases and cyclic AMP response element-binding protein. ME significantly increased pre- and post-synapse formation, acetylcholine synthesisation, neuronal cell differentiation, neurite outgrowth and neuronal cell proliferation in the mouse hippocampus. Furthermore, ME significantly increased latency time in the passive avoidance task (P< 0·001) and recognition time of novel objects in the object recognition test (P< 0·05), indicating improvements in learning and memory. Taken together, these data suggest that ME exhibits a memory-enhancing effect via up-regulation of NGF.

Nuclear choline acetyltransferase activates transcription of a high-affinity choline transporter.

Choline acetyltransferase (ChAT) synthesizes the neurotransmitter, acetylcholine, at cholinergic nerve terminals. ChAT contains nuclear localization signals and is also localized in the nuclei of neural and non-neuronal cells. Nuclear ChAT might have an as yet unidentified function, such as transcriptional regulation. In this study, we investigated the alteration of candidate gene transcription by ChAT. We chose high affinity choline transporter (CHT1) and vesicular acetylcholine transporter (VACHT) as candidate genes, which function together with ChAT in acetylcholine production. Using SH-SY5Y human neuroblastoma cells stably expressing wild-type human ChAT, we found that overexpressed ChAT enhanced transcription of the CHT1 gene but not the VACHT gene. In contrast, nuclear localization signal disrupted, and catalytically inactive mutant ChATs could not induce, CHT1 expression. Additionally, ChAT did not alter CHT1 expression in non-neuronal HEK293 cells. Our results suggest that ChAT activates the transcription of selected target genes in neuronal cells. Both enzymatic activity and nuclear translocation of ChAT are required for its transcriptional enhancement.

The vesicular glutamate transporter VGLUT3 synergizes striatal acetylcholine tone.

Three subtypes of vesicular transporters accumulate glutamate into synaptic vesicles to promote its vesicular release. One of the subtypes, VGLUT3, is expressed in neurons, including cholinergic striatal interneurons, that are known to release other classical transmitters. Here we showed that disruption of the Slc17a8 gene (also known as Vglut3) caused an unexpected hypocholinergic striatal phenotype. Vglut3(-/-) mice were more responsive to cocaine and less prone to haloperidol-induced catalepsy than wild-type littermates, and acetylcholine release was decreased in striatum slices lacking VGLUT3. These phenotypes were associated with a colocalization of VGLUT3 and the vesicular acetylcholine transporter (VAChT) in striatal synaptic vesicles and the loss of a synergistic effect of glutamate on vesicular acetylcholine uptake. We propose that this vesicular synergy between two transmitters is the result of the unbalanced bioenergetics of VAChT, which requires anion co-entry for continuing vesicular filling. Our study reveals a previously unknown effect of glutamate on cholinergic synapses with potential functional and pharmacological implications.

Analysis of Caenorhabditis elegans acetylcholine synthesis mutants reveals a temperature-sensitive requirement for cholinergic neuromuscular function.

In Caenorhabditis elegans, the cha-1 gene encodes choline acetyltransferase (ChAT), the enzyme that synthesizes the neurotransmitter acetylcholine. We have analyzed a large number of cha-1 hypomorphic mutants, most of which are missense alleles. Some homozygous cha-1 mutants have approximately normal ChAT immunoreactivity; many other alleles lead to consistent reductions in synaptic immunostaining, although the residual protein appears to be stable. Regardless of protein levels, neuromuscular function of almost all mutants is temperature-sensitive, i.e., neuromuscular function is worse at 25° than at 14°. We show that the temperature effects are not related to acetylcholine release, but specifically to alterations in acetylcholine synthesis. This is not a temperature-dependent developmental phenotype, because animals raised at 20° to young adulthood and then shifted for 2 h to either 14° or 25° had swimming and pharyngeal pumping rates similar to animals grown and assayed at either 14° or 25°, respectively. We also show that the temperature-sensitive phenotypes are not limited to missense alleles; rather, they are a property of most or all severe cha-1 hypomorphs. We suggest that our data are consistent with a model of ChAT protein physically, but not covalently, associated with synaptic vesicles; and there is a temperature-dependent equilibrium between vesicle-associated and cytoplasmic (i.e., soluble) ChAT. Presumably, in severe cha-1 hypomorphs, increasing the temperature would promote dissociation of some of the mutant ChAT protein from synaptic vesicles, thus removing the site of acetylcholine synthesis (ChAT) from the site of vesicular acetylcholine transport. This, in turn, would decrease the rate and extent of vesicle-filling, thus increasing the severity of the behavioral deficits.

Phenotypical and Myopathological Consequences of Compound Heterozygous Missense and Nonsense Variants in SLC18A3.

BACKGROUND: Presynaptic forms of congenital myasthenic syndromes (CMS) due to pathogenic variants in SLC18A3 impairing the synthesis and recycling of acetylcholine (ACh) have recently been described. SLC18A3 encodes the vesicular ACh transporter (VAChT), modulating the active transport of ACh at the neuromuscular junction, and homozygous loss of VAChT leads to lethality. METHODS: Exome sequencing (ES) was carried out to identify the molecular genetic cause of the disease in a 5-year-old male patient and histological, immunofluorescence as well as electron- and CARS-microscopic studies were performed to delineate the muscle pathology, which has so far only been studied in VAChT-deficient animal models. RESULTS: ES unraveled compound heterozygous missense and nonsense variants (c.315G>A, p.Trp105* and c.1192G>C, p.Asp398His) in SLC18A3. Comparison with already-published cases suggests a more severe phenotype including impaired motor and cognitive development, possibly related to a more severe effect of the nonsense variant. Therapy with pyridostigmine was only partially effective while 3,4 diaminopyridine showed no effect. Microscopic investigation of the muscle biopsy revealed reduced fibre size and a significant accumulation of lipid droplets. CONCLUSIONS: We suggest that nonsense variants have a more detrimental impact on the clinical manifestation of SLC18A3-associated CMS. The impact of pathogenic SLC18A3 variants on muscle fibre integrity beyond the effect of denervation is suggested by the build-up of lipid aggregates. This in turn implicates the importance of proper VAChT-mediated synthesis and recycling of ACh for lipid homeostasis in muscle cells. This hypothesis is further supported by the pathological observations obtained in previously published VAChT-animal models.

An 8-Week Administration of Bifidobacterium bifidum and Lactobacillus plantarum Combined with Exercise Training Alleviates Neurotoxicity of Aß and Spatial Learning via Acetylcholine in Alzheimer Rat Model.

This study aimed to determine the effects of 8 weeks of an administration of Bifidobacterium bifidum and Lactobacillus plantarum combined with exercise training on neurotoxicity of Aß, spatial learning, acetylcholine (ACH), and vascular endothelial growth factor (VEGF) in Alzheimer rats. Twenty-five Wistar rats were randomly divided into 5 groups (n = 5 in each): (1) healthy control (control), (2) Alzheimer disease (AD), (3) AD with treadmill exercise (AD + Exe), (4) AD with probiotic (combined administration of Bifidobacterium bifidum and Lactobacillus plantarum) treatment (AD + Pro), and (5) AD with treadmill exercise and probiotic treatment (AD + Exe + Pro). AD was induced by intra-cerebroventricular injection of Aß1-42 peptide. Then, the training groups exercised on treadmill for 8 weeks, 5 days per weeks. The rats were treated daily with probiotic supplements via gavage for 8 weeks. The Morris water maze (MWM) test was administered to measure spatial learning. Then, the animals were sacrificed and Vegf and ACH were analyzed using the qPCR and immunohistochemistry (IHC) methods, respectively. Results showed that the ß-amyloid plaques were significantly increased in the brains of the AD group compared with the control group (p < 0.001). The combined use of probiotics and exercise training significantly increased the time spent in the target quadrant after removing the platform, compared with the AD group in the Morris water maze test (p < 0.001). Crystal violet analysis showed that sole (p < 0.01) and combined exercise training and probiotic supplementation (p < 0.001) significantly reduced the number of dead cells in the brains of rats compared with the AD group. AD significantly decreased Vegf mRNA and ACH in the CA1 area of the hippocampus (p < 0.001). However, mono and combined therapy (exercise and probiotics) significantly increased ACH in the rats' brain compared with the AD group. Overall, 8 weeks of an administration of Bifidobacterium bifidum and Lactobacillus plantarum combined with exercise training can improve spatial learning impairment in the AD rats. Exercise and probiotics seem to offer potential benefits to AD patients by upregulating ACH.